Moreover, these effects can be inhibited by the corresponding miR inhibitor. In addition, we found that high levels of mir-885-3p could inhibit the AKT/NFκB signaling pathway, upregulate the GRE plasmid activity and GR level, and downregulate the level of inflammatory factors of OFs. Moreover, the improvement of glucocorticoid sensitivity by mir-885-3p transmitted by SI-exo can also be inhibited by the AKT/NFκB agonist. Finally, through the in vivo experiment of the GO mouse model, we further determined the relationship between exosomes’ mir-885-3p sequence, AKT/NFκB signaling pathway, and glucocorticoid sensitivity. As a conclusion, plasma exosomes deliver mir-885-3p and inhibit the AKT/NFκB signaling pathway to improve the glucocorticoid sensitivity of OFs. Exosome mir-885-3p can be used as a biomarker to determine the sensitivity of ivGCs in GO patients.Graves ophthalmopathy (GO), a manifestation of Graves’ disease, is an organ-specific autoimmune disease. Intravenous glucocorticoid therapy (ivGCs) is the first-line treatment for moderate-to-severe and active GO. However, ivGCs is only effective in 70%–80% of GO patients. Insensitive patients who choose 12-week ivGCs not only were delayed in treatment but also took the risk of adverse reactions of glucocorticoids. At present, there is still a lack of effective indicators to predict the therapeutic effect of ivGCs. Therefore, the purpose of this study is to find biomarkers that can determine the sensitivity of ivGCs before the formulation of treatment, and to clarify the mechanism of its regulation of ivGCs sensitivity. This study first characterized the miRNA profiles of plasma exosomes by miRNA sequencing to identify miRNAs differentially expressed between GO patients with significant improvement (SI) and non-significant improvement (NSI) after ivGCs treatment. Subsequently, we analyzed the function of the predicted target genes of differential miRNAs. According to the function of the target genes, we screened 10 differentially expressed miRNAs. An expanded cohort verification showed that compared with NSI patients, mir-885-3p was upregulated and mir-4474-3p and mir-615-3p were downregulated in the exosomes of SI patients. Based on statistical difference and miRNA function, mir-885-3p was selected for follow-up study. The  Graves ophthalmopathy (GO) is an organ-specific autoimmune disease associated with Graves’ disease . 25%–50%IvGCs is the first-line treatment for moderate-to-severe active GO. For mild GO, ivGCs is also recommended when the quality of life is seriously affected . In otheStudies found that the clinical signs of GO patients , the durThe biomarkers that can predict the sensitivity of ivGCs in GO patients mainly include proteins, nucleic acids, and lipids. It is generally believed that the nucleotide sequence change of GR can change the sensitivity of glucocorticoid sensitivity. However, this phenomenon seems not to be found in GO patients treated with ivGCs. A study of 58 GO patients treated with ivGCs found that the 3 GR gene polymorphisms, ER22/23EK, N363s, and BCL1 do not influence the therapeutic effect of steroids . Li founhttp://www.exorbase.org/exoRBaseV2/toIndex) and exocorta (http://exocarta.org/index.html) were established. However, serum miRNAs are fragile and very sensitive to RNA enzymes in the air, and its level will fluctuate with physical factors. Under the protection of the double-layer membrane structure, plasma exosome miRNAs remain stable without degradation. More importantly, exosome miRNAs can be easily and stably extracted even in long-term frozen samples. Plasma exosome miRNAs may be helpful to determine the sensitivity of ivGCs.Exosomes are 30–150-nm membrane vesicles secreted by almost all types of human cells. In 1983, exosomes were first found in sheep reticulocytes. After nearly 40 years of research, exosome detection technologies are becoming more and more mature, and independent databases of exosomes such as exoRBase . The particle size distribution and concentration of exosomes were detected by a NOT analyzer . Exosome morphology was observed by transmission electron microscopy . Exosome protein concentrations were determined by BCA protein quantification . Finally, exosome-specific marker proteins CD9, CD81, and TSG101 were detected by Western blot.Three patients in both SI and NSI groups were randomly selected for exosome miRNA sequencing. Sequencing data were analyzed, and the differentially expressed exosome miRNAs were defined as miRNAs with p value <0.05 and log2(fold change) ≠ 0.The target genes of miRNA identified in differential exosomes were predicted by miRanda and RNAhyThe possible sites of mir-885-3p predicting AKT intervention were queried in the miRNA database. Two pairs of DNA single strands Table S3The GO animal model was established by Balb/c female mice aged 8–10 weeks. The establishment method of the model group was the same as our previous study , and theOther mice in the model group were randomly divided into 6-week treatment group, 9-week treatment group, 12-week treatment group, and corresponding control group. In the first 6 weeks of treatment, mice in the treatment group were given methylprednisolone 1 mg/kg intraperitoneal injection once a week. From the 7th week, the dose of methylprednisolone was adjusted to 0.5 mg/kg. The control group was given the same amount of normal saline. The mice were sacrificed at the 6th, 9th, and 12th weeks of treatment, and their plasma and orbital tissue were collected for analysis. All mice were provided by the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University, raised in an environment free of specific pathogens and operated in accordance with the humane animal care standards stipulated by Harbin Medical University. Animal experiment was reviewed by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University, China (no. KY2017-110).2-humidified environment. The cells were identified by immunofluorescence.GO mouse extraocular muscle was obtained under aseptic conditions, and primary OFs were cultured. The OFs were cultured in Dulbecco’s modified Eagle medium  with 10% fetal bovine serum  and incubated in a constant 37°C, 5% CO500 μg purified exosome suspension was taken and diluted to a volume of 500 μl with sterile 1× PBS. According to the instructions, exosomes were labeled with PKH26 staining kit . After staining, the concentration of the stained exosome suspension was quantified by the BCA method , and then aseptic 1× PBS adjusted the exosomes to the appropriate concentration.4 OFs were spread to a 24-well plate containing sterile slides. Cells were adjusted to a bottling rate of 50%, and a complete culture medium containing 25 μg/ml PKH26-labeled exosomes was added for 24 h. After PBS cleaning, fixation was performed with 4% formaldehyde solution, room temperature peroxidation with 0.5% Triton X-100 solution , cytoskeleton staining with FITC working solution , and nucleus staining with DAPI . Finally, they were observed and photographed by a fluorescence microscope.5 × 105 per vial. 10-μl miRNA plasmids . Protein lysates were transferred to the PVDF membrane by 8% SDS-PAGE . The PVDF membrane was cleaned with TBST and sealed with 5% skim milk powder for 2 h. The cut film was incubated overnight in the primary antibody dilution solution and 2 h in the secondary antibody dilution solution. Finally, the film was scanned. The main antibodies used included HRP-binding GAPDH monoclonal antibody , AKT polyclonal antibody , NFκB P65 polyclonal antibody , Rabbit Anti-phosphorylated NFκB P65 (Ser276) Antibody , Rabbit Anti-phosphorylated AKT (Ser473) antibody , IL-1α polyclonal antibody , ICAM1 polyclonal antibody , CD9 monoclonal antibody , TSG101 polyclonal antibody , CD81 monoclonal antibody , Goat Anti-mouse IgG (H+L) , Goat anti-rabbit IgG (H+L) , and glucocorticoid receptor polyclonal antibody .TRIzol  extracted total RNA from OFs, exosomes, and tissues. 1 µg RNA was reversed transcribed using the transcript first-strand cDNA synthesis Kit . Thermal cycling conditions were as follows: pre-denaturation at 95°C for 3 min, 95°C for 20 s, and 60°C for 45 s for 40 cycles. Fluorescence was measured at the end of each cycle. Primers were synthesized by Shanghai Tianhao Biotechnology Co., Ltd., China. Detailed sequences information of primers are shown in The OFs transfected with pGRE-luc  and pRL-TK  were incubated with lysate, and 300-μl samples were taken into the chemiluminescence detection tube. 500 μl 1× firefly luciferase reaction solution  was added to the reaction tube. The Junior LB 9509 chemiluminescence detector detected the luminescence value for 10 s. 500 μl 1× Aquin luciferase reaction solution  was added and gently blown and mixed. The luminescence value was detected by a Junior LB 9509 chemiluminescence detector for 10 s. Relative luciferase activity was calculated based on the two luminescence obtained in each group.In this study, three patients in the SI and NSI groups were randomly selected to extract plasma exosomes . Transmission electron microscopy, Western blot, and nanoparticle tracking analysis (NTA) were used to verify that the extract was plasma exosome. Transmission electron microscopy showed that there was no significant difference in the morphology of plasma exosomes between SI and NSI, with a diameter of about 100 nm and a typical concave disc-like structure  and 4 miRNAs downregulated (miR-615-3p/miR-4474-3p/miR-143-5p/miR-5581-3p) in the SI group. The levels of the above miRNA in plasma exosomes of the remaining 11 patients were detected by PCR. Compared with the NSI group, the expression of mir-885-3p was highly expressed (p = 0.001), and mir-4474-3p (p < 0.05) and mir-615-3p (p < 0.05) were low expressed . Thus, mir-885-3p was selected for follow-up mechanism research.The above 12 target genes correspond to 10 candidate miRNAs. Compared with the NSI group, there were 6 miRNAs upregulated  (p < 0.01), and the protein (p < 0.01) and mRNA (p < 0.001) levels of inflammatory factors interleukin-1 (IL-1) and intercellular cell adhesion molecule-1 (ICAM-1) were significantly downregulated  with OFs for 24 h. The merged image of immunofluorescence showed that the plasma exosomes were endocytosed by OFs  (p < 0.001). The same trend results also appeared in the NSI-exo group. This indicates that the miR inhibitor can inhibit well the upregulation of miR-885-3p by exosomes (p < 0.001) (p < 0.001), and inflammatory factor IL-1 and ICAM-1 protein levels (p < 0.001) and mRNA levels (p < 0.01) were significantly upregulated  (p<0.001), and inflammatory factors were significantly downregulated  (p < 0.001)  and pNFκB (p < 0.001) levels which were significantly downregulated , but the GRE gene plasmid activity was significantly decreased after the addition of SC79 (p < 0.001) (p < 0.001), and the protein and mRNA levels of inflammatory factors IL-1 and ICAM-1 were lower than those in the Mir-NC group (p < 0.001). After the addition of SC79, the protein and mRNA levels of GR, IL-1, and ICAM1 showed an opposite trend compared with the mir-885-3p group  (p < 0.001). The same trend results also appeared in the NSI-exo group , week 9 (p < 0.01), and week 12 (p < 0.001) of the treatment course (p < 0.001), while AKT (p < 0.001) and NFκB (p < 0.001) were downregulated , we predicted that mir-885-3p targeted the regulation of AKT. As a classical molecule of the chemokine signaling pathway, AKT regulates the expression of downstream protein NFκB  of mice were insensitive to glucocorticoid, while at the 9th and 12th weeks, the proportion of insensitive mice decreased to 3/14 and 3/12, respectively. The proportion of glucocorticoid-insensitive mice was similar at weeks 9 and 12. This result is consistent with the report by Guia that the sensitivity of ivGCs can be judged according to the response of GO patients during 6–8 weeks of treatment , which ain vitro experiment. We confirm that the changes of exosome mir-885-3p sequence, AKT/NFκB, and glucocorticoid sensitivity still existed in vivo.Due to the large amount of plasma required for extraction of exosomes and the small body weight of mice, it cannot ensure that mice can still survive when enough plasma is collected. Therefore, the existing technique in this study was unable to detect baseline plasma exosome mir-883-3p sequence levels in mice. We supported the conclusion of this study by detecting the level of plasma exosome mir-883-3p sequence in mice at the 6th, 9th, and 12th weeks of treatment. It was found that compared with the insensitive group, plasma exosome mir-885-3p sequence and GR level were upregulated, and AKT and NFκB levels were downregulated in the sensitive group after glucocorticoid treatment, which was consistent with the results In conclusion, this study provided the effective plasma exosome miRNA expression profile of GO patients by miRNA sequencing and searched stable and easily detected circulating biomarkers of ivGCs sensitivity. Meanwhile, the mechanism of exosome mir-885-3p regulating the sensitivity of ivGCs was elucidated: SI-Exo transfer mir-885-3p targeting the AKT/NFκB signaling pathway improves the ivGCs sensitivity of GO patients. Plasma exosome mir-885-3p is expected to become a reliable and feasible biomarker to predict GO patients’ sensitivity of ivGCs. It provides a scientific basis for the selection of treatment methods for GO patients and is of great significance to ensure the good prognosis of patients.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE190515.The original contributions presented in the study are publicly available. These data can be found here: The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University, China (no. KY2016-046). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University, China (no. KY2017-110).HQ conceived the idea of this manuscript. JS performed the experiment. JW, YZ, JjL, JL, JY, MG, and JH collected the data and performed the data analysis. JS wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Fund of China (grant nos. 81872560 and 82073491).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In most cases, the carcinogenesis of colorectal cancer (CRC) follows the normal-adenoma-carcinoma (N-A-C) sequence. In this study, we aimed to identify the key proteins in the N-A-C sequence.  Differentially expressed proteins (DEPs) in normal, adenoma, and carcinoma tissues were identified using the Tandem Mass Tag- (TMT-) based quantitative proteomics approach. The landscape of proteomic variation in the N-A-C sequence was explored using gene set enrichment analysis (GSEA) and Proteomaps. Key proteins in the N-A-C sequence were identified, verified, and validated based on our proteomic data, external proteomic data, and external transcriptomic data in the ProteomeXchange, CPTAC, GEO, and TCGA databases. The prognostic value of the key proteins in our database was evaluated by univariate and multivariate Cox regression analysis. The effects of the key proteins on adenoma organoids and colorectal cancer cells were explored in functional studies.  Based on our proteomic profiles, we identified 1,294 DEPs between the carcinoma (CG) and normal (NG) groups, 919 DEPs between the adenoma group (AG) and NG, and 1,030 DEPs between the CG and AG. Ribosome- and spliceosome-related pathways were mainly enriched in the N-A process. Extracellular matrix- and epithelial-mesenchymal transition- (EMT-) related pathways were mainly enriched in the A-C process. RRP12 and SERPINH1 were identified, verified, and validated as candidate key proteins in the N-A and A-C processes, respectively. Furthermore, RRP12 and SERPINH1 knockdown impeded the viability and proliferation of adenoma organoids. SERPINH1 was validated as a risk factor for disease-free survival (DFS) based on the TCGA and our database, whereas RRP12 did not show prognostic value. SERPINH1 knockdown was accompanied by EMT-related protein variation, increased apoptosis, and reduced proliferation, invasion, and migration of CRC cells in vitro.  RRP12 and SERPINH1 may play an important role in the N-A and A-C processes, respectively. Furthermore, SERPINH1 showed favorable prognostic value for DFS in CRC patients. We speculate that SERPINH1 might promote not only the A-C process but also the development of CRC. Colorectal cancer (CRC) is the second leading cause of cancer deaths, accounting for approximately 9.2% of all cancer deaths annually . ApproxiBased on the major principle of carcinogenesis, the majority of studies on colorectal cancer have employed a genomic analysis approach to explore the N-A-C sequence –11. SomeThe low rate of carcinogenesis from adenoma indicates that the driver events of the process of transformation from normal mucosa to adenoma (N-A) may be different from that from adenoma to carcinoma (A-C). Furthermore, in the N-A-C sequence, the “mutational hits” in the N-A process are different from those in the A-C process . Hence, Normal, adenoma, and carcinoma tissues were obtained from 23 patients recruited in the Division of Colorectal Surgery, Department of General Surgery, Peking Union Medical College Hospital (China). The clinical and pathological characteristics of the patients are listed in Supplementary µg) in 8 M urea were reductive with dithiothreitol (DTT) and were alkylated with iodoacetamide (IAA). Then the proteins were dissociated with Trypsin/Lys-C, and peptide labeling was performed using the TMT Kit. The TMT-labeled peptides were subjected to high-performance liquid chromatography (HPLC) analysis followed by analysis with a directly interfaced Thermo Orbitrap Fusion mass spectrometer (Thermo Scientific). Protein identification was performed using Proteome Discoverer 2.2 software (Thermo Scientific) with the SEQUEST search engine. Only proteins with a false discovery rate (FDR) < 0.01 and unique peptide ≥2 qualified for the further analysis. Relative protein quantification was performed using the TMT-6plex method. All methods were performed as previously described [http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier: PXD017068 and PXD023899 [Lysate proteins (100 escribed . The mashttps://www.ncbi.nlm.nih.gov/geo/). The gene expression matrices of the GSE 117606, GSE 50014, and GSE 50015 datasets were downloaded for coanalysis with the proteomic data. Differentially expressed genes (DEGs) between normal tissues and adenoma tissues and between adenoma tissues and carcinoma tissues in the three datasets were identified using the GEO2R tool (https://www.ncbi.nlm.nih.gov/geo/geo2r/) following the criteria of adjusted P value < 0.05 and |log2 fold change (FC) ratio| >0.3. GSE 20916 and GSE 41567 datasets were used to validate the key proteins.The gene expression profiles and corresponding clinical information were acquired from the GEO database  and anti-RRP12  for 24 h. On the second day, the sections were incubated with second antibodies  and stained with DAB . IHC scores were calculated as the product of stain-positive cell score and the staining intensity score. The stain-positive cell score was determined as follows: 1 = <25% cells, 2 = 25%–50% cells, 3 = 50%–75% cells, and 4 = >75% cells. Staining intensity was determined as follows: 0 = no staining, 1 = low-level staining, 2 = intermediate-level staining, and 3 = high-level staining. High and low IHC scores were defined as 9–12 and 0–8, respectively.Paraffin-embedded normal, adenoma, and carcinoma tissue samples were cut into 4 http://jvenn.toulouse.inra.fr/app/example.html) [https://cran.r-project.org/bin/windows/base/) and the “Pheatmap” package. Gene set enrichment analysis (GSEA) software and the molecular signature database, MsigDB, were downloaded from the Broad Institute website (http://www.broadinstitute.org/gsea/index.jsp) [http://www.Proteomaps.net/) were used to show the function of the differentially expressed proteins (DEPs) using the polygon module based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways gene classification [https://string-db.org/) [http://gepia.cancer-pku.cn/) and UALCAN are online databases including colorectal normal and cancer gene expression data, corresponding with the clinicopathological and prognostic information of the Cancer Genome Atlas (TCGA) [Common proteins in the three replicates were identified using a Venn diagram webtool (le.html) . Hierarcdex.jsp) . The canfication . The sizdb.org/) . We thendb.org/) , 23. Thedb.org/) . GEPIA (s (TCGA) , 26. TheFresh adenoma tissue was cut into small pieces, washed with PBS, and dissociated in TrypLE  to achieve cell suspension. We then used Sato's method to prepare the culture medium, which was changed every 3 days . The orgSW480 cells were seeded 1 day in advance to ensure that the cell confluence reached 70%–80% before transfection. According to the instructions, Opti-MEM  was used to dilute RNAiMAX , SERPINH1-siRNA1, siRNA2, and siRNA3 , respectively. The diluted RNAiMAX was mixed with each siRNA dilution and the mixed dilution was added to the cell culture medium after standing still for 5 min. The final concentration of SERPINH1-siRNA1, siRNA2, and siRNA3 was 50 nM, respectively. After incubating at 37°C for 48 hours, the cells were harvested for subsequent experiments.Cell Recovery Solution  was used to dissociate Matrigel. Organoids pellet was obtained after centrifugation, and then the centrifugation was dissociated by TrypLE  for 10 min at 37°C. SERPINH1-siRNA2 was diluted according to the above description and the final concentration was 50 nM. After centrifugation, the organoids were incubated at 37°C for 4 hours. The organoids were collected and seeded in Matrigel for further incubation after removing the supernatant. After 48 hours, they were collected for subsequent experiments.After transfection with siRNAs, organoids were fixed with 4% paraformaldehyde and blocked. Organoids were then incubated with anti-Ki-67  at 4°C overnight. Then, cells were incubated with Alexa Fluor 488 conjugated Goat anti-Rabbit IgG  at 4°C overnight. Finally, the pictures were captured using confocal microscope .μl of the ddPCR reaction mixture.Total RNA was extracted from the SW480 cell lines using TRIzol reagent (Thermo Scientific) according to the manufacturer's instructions. RNA was reversely transcribed into cDNA using the QuantiTect Reverse Transcription Kit  according to the manufacturer's instructions. Real-time quantitative PCR was performed using SYBR-Green , and the expression levels were normalized to that of GAPDH. The quantity of DNA in adenoma organoids was evaluated by ddPCR. Approximately 200 ng DNA extracted from each organoids was analyzed in 14.5   GAPDH   RRP12   SERPINH1 The qRT-PCR was performed using the following primers: SW480 cells were seeded in 96-well plates . The proliferation ability of cells was evaluated using the Cell Counting Kit-8  according to the manufacturer's protocols. After culturing for 2–3 h, the absorbance in each well was measured at a wavelength of 450 nm using a multimicroplate test system.® 3D Reagent  was used to dissolve Matrigel. The adenoma organoids suspension was acquired and then was equally distributed to opaque-walled multiwell plates . A volume of CellTiter-Glo® 3D Reagent equal to the volume of cell culture medium was added to each well and then the content was incubated for 30 min at room temperature. Finally, total ATP content of each well determines the cell viability.The adenoma organoids transfected with SERPINH1-siRNA (refer to the method: adenoma organoids siRNA transfection) was used to detect cell viability. The CellTiter-GloFor cell apoptosis analysis, cells were incubated for 48 h following transfection and then processed with a FITC Annexin-V Apoptosis Detection Kit  according to the manufacturer's instructions and analyzed by flow cytometry. Data were analyzed using FlowJo 10.7.1 software.4 cells/well were resuspended in 100 μl fetal bovine serum-free medium in the upper chamber  of a Transwell system. The lower chamber was filled with 0.6 ml medium supplemented with 10% FBS. After incubation for 48 h at 37°C, the invasive cells were fixed with 100% methanol and stained with 0.5% crystal violet before counting under an inverted microscope. For the migration assay, 2.5 × 104 cells were plated in uncoated Transwell upper chambers. The number of cells that migrated across the membrane was estimated under an inverted microscope  at 20× magnification.Cell invasion and migration abilities were evaluated using Transwell assays. For the invasion assay, Matrigel chambers  were used according to the manufacturer's instructions. A total of 2.5 × 10β-catenin , NF-κB p65 , Twist , Slug , GSK-3β , phospho-GSK-3β , N-cadherin , E-cadherin , MMP9 , and MMP14 . The membrane was then washed and incubated with a horseradish peroxidase-conjugated secondary antibody.Protein concentration was determined using BCA Protein Assay Kits  according to the manufacturer's instructions. All protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and incubated overnight at 4°C with primary antibodies for the detection of the following: SERPINH1 , P < 0.05 and |log2 FC ratio| >0.3) identified in paired comparisons of NG, AG, and CG was evaluated using Student's t-test based on the grouped proteomic abundance and depicted volcano plot. Receiver operating characteristic (ROC) curves were generated to validate the discrimination value of RRP12 and SERPINH1. The Kaplan–Meier method was used for survival analysis. Univariate and multivariate Cox regression analyses were used to identify independent prognostic factors. The criterion for inclusion in the multivariate Cox regression analysis was P < 0.1. P < 0.05 was considered to indicate statistical significance.Statistical analysis was performed using GraphPad Prism 8.0.1 . The significance of DEPs . More DEPs were upregulated than downregulated in the N-A, A-C, and N-C processes.The workflow of our study is shown in Supplementary It is widely accepted that the N-A and A-C processes represent two phases of colorectal carcinogenesis driven by different events in a sequential manner . TherefoIn the GSEA, alternative mRNA splicing, regulation of mRNA processing, ribosome biogenesis, and the spliceosome pathway were found to be enriched by comparison of the proteomic profiles of NG and AG . ExtraceThe enrichment analysis of DEPs in the two phases was visualized through the construction of a Proteomap, in which each protein is presented by a small polygon tile. The area of tile reflects the protein abundance ratio of the compared groups. This analysis showed that the DEPs between NG and AG were primarily enriched in spliceosome-, RNA transport-, ribosome-, and chromosome-related pathways . The DEPThe results of the GSEA and Proteomap analysis were similar and both indicated the entirely different progression modes for the N-A and A-C processes.P value < 0.05 and log2 FC ratio >0.3).Due to the different proteomic changes of N-A and A-C processes, we separated the two parts and performed integration analysis between the proteomic profiles of each part and the external transcriptomic datasets. Three gene expression profiles  were downloaded from the GEO database as external transcriptomic datasets to identify upregulated DEGs following the same screen criteria that were applied to our dataset  group showed significantly decreased DFS compared with the low SERPINH1 IHC score (<9 points) group  for the N-A and A-C processes. In the verification step, we confirmed RRP12 and SERPINH1 as the key proteins in the N-A and A-C processes, respectively, by using cytoHubba to rank hub proteins and an external proteomic database to evaluate the expression level. In the validation phase, we confirmed the discrimination value of RRP 12 and SERPINH1 in our own dataset and databases. Furthermore, the two proteins were positively correlated with the viability and proliferation of adenoma organoids. SERPINH1 was identified as a risk factor for DFS in TCGA and our own database. Finally, based on the results of Western blot and functional studies, we speculated that SERPINH1 may correlate with EMT and promote the development of cancer. Thus, our study elucidates the proteomic variation enrichment in the N-A and A-C processes and clarifies the evolution of the N-A-C sequence in protein level. Furthermore, the majority of previous studies designed to identify biomarkers of the N-A-C sequence were focused on the discrimination value. In the current study, a key protein, SERPINH1, in the A-C process was screened out, and it was found to be of discriminatory and prognostic value. Therefore, SERPINH1 may become a candidate biomarker and a potential therapeutic target for clinical application. These findings were further supported by the results of our functional studies on both and cancer cell lines adenoma organoids.We found that ribosome- and spliceosome-related events were significantly enriched in the N-A process. Ribosomes are important organelles for protein synthesis and are composed of ribosomal proteins (RPs) and RNAs (rRNAs). Although the main function of RPs is structural, the important role of RPs in tumorigenesis cannot be ignored. Leonart et al. reported that some RPs are associated with oncogene activation, and dysfunction of RPs may contribute to tumorigenesis directly . Xu and We found that ECM- and EMT-related pathways were significantly enriched in the A-C process. The ECM is a crucial structure in the tumor microenvironment. Under normal conditions, the primary function of the ECM is to maintain homeostasis and orchestrate tissue repair in response to injury or damage . In the Some limitations of this study should be noted. First, the results are mainly based on proteomic analysis and functional studies, and the potential mechanisms underlying the roles of RRP12 and SERPINH1 in the N-A-C sequence remain to be elucidated. An in-depth exploration of the mechanism by which SERPINH1 promotes cancer development is also warranted. Second, our analysis was performed on a small number of clinical samples and the prognostic ability of the candidate key proteins identified in this study requires validation in a large cohort. Third, this study was based on only proteomic and transcriptomic analyses and the events that drive the N-A-C sequence occur at the genetic level. Our analysis showed that metabolism-related pathways were obviously enriched in the A-C process. Therefore, a combination of genomics and metabolomics approaches may provide a more comprehensive understanding of the N-A-C sequence.There are two significances of this study: (1) This study indicated that RRP12 and SERPINH1 were the key proteins in the N-A process and the A-C process, respectively. This finding would contribute to target prevention of the carcinogenesis in CRC and then accelerate secondary prevention of CRC precisely. (2) SERPINH1 might play a key role in both the A-C process and the development of CRC. Moreover, SERPINH1 could be secreted into extracellular space. Hence, SERPINH1 could potentially be the noninvasive biomarker reflecting the adenoma and carcinoma progression.In conclusion, we comprehensively analyzed the proteomic variations in the N-A-C sequence. Ribosome- and spliceosome-related pathways were mainly enriched in the N-A process, whereas ECM- and EMT-related pathways were mainly enriched in the A-C process. RRP12 and SERPINH1 may play an important role in the N-A and A-C processes, respectively. Furthermore, SERPINH1 showed favorable prognostic value for DFS in CRC patients. We speculate that SERPINH1 might promote not only the A-C process but also the development of CRC.


ObjectiveIn this study, we aimed to determine the association between anti-thyroid peroxidase (anti-TPO) antibody and dyslipidemia in subclinical hypothyroidism (SCH).Materials and methodsWe conducted a cross-sectional case-control study in the department of medicine at a tertiary care teaching hospital in central India. The study consisted of 150 patients (75 cases and 75 controls) who fulfilled the inclusion and exclusion criteria.ResultsThe study showed that serum cholesterol was high in 23.1% of cases in the negative anti-TPO antibody SCH sub-group and 88.7% of cases in the positive anti-TPO antibody SCH sub-group (p<0.001). Serum triglyceride (TG) levels were high in 61.5% of cases in the negative anti-TPO antibody SCH sub-group and 90.3% of cases in the positive anti-TPO antibody SCH sub-group (p=0.008). Serum high-density lipoproteins (HDL) were low in 15.4% of cases in the negative anti-TPO antibody SCH sub-group and 24.2% of cases in the positive anti-TPO antibody SCH sub-group (p=0.490). Serum low-density lipoproteins (LDL) were high in 61.5% of cases in the negative anti-TPO antibody SCH sub-group and 90.3% of cases in the positive anti-TPO antibody SCH sub-group (p=0.008). Serum very-low-density lipoproteins (VLDL) were high in 84.6% of cases in the negative anti-TPO antibody SCH sub-group and 83.9% of cases in the positive anti-TPO antibody SCH sub-group (p=0.947). Based on our findings, 82.8% of participants with negative anti-TPO antibodies had normal serum LDL levels, while 11.1% of participants with positive anti-TPO antibodies had normal LDL levels (p<0.001). The study also showed that elevated serum LDL was present in 17.2% of participants with negative anti-TPO antibody levels and 88.9% of participants with positive anti-TPO antibodies (p<0.001). The study showed a correlation coefficient of 0.0432 between serum TG and anti-TPO antibody levels (p<0.001).ConclusionOur findings showed an increased incidence of dyslipidemia in SCH patients with positive anti-TPO antibodies. SCH with positive anti-TPO antibody is significantly associated with elevated serum total cholesterol (TC) levels, serum TG levels, and serum LDL levels. Hence, early screening and diagnosis of dyslipidemia are crucial to prevent cardiovascular morbidity and mortality in SCH patients with positive anti-TPO antibodies. The American Thyroid Association defines subclinical hypothyroidism (SCH) as a condition with elevated thyroid-stimulating hormone (TSH) levels accompanied by thyroid hormone levels in the normal range. However, this definition is only applicable when thyroid function has been stable, the hypothalamic-pituitary-thyroid axis is normal, and in the absence of any recent or ongoing severe illness . The impLipid profile changes in overt hypothyroidism are well established; however, the results regarding lipid profile changes in SCH have been controversial. Althaus et al., in their study of SCH, found a 10% increase of serum low-density lipoprotein (LDL) (p<0.01) and a 10% decrease of high-density lipoprotein (HDL) (p<0.05) while there were no significant changes in total cholesterol (TC) and serum triglyceride (TG) levels . HoweverAs mentioned above, there is scant regional data on the relationship between anti-TPO antibodies and dyslipidemia in SCH. For early screening and diagnosis of patients at risk of cardiovascular events, it is crucial to study the association between TPO antibodies and dyslipidemia in SCH. In this study, we aimed to analyze the association between dyslipidemia and anti-TPO antibodies in patients with SCH.Study design and settingThis was a cross-sectional case-control study conducted in the outpatient section of the department of internal medicine at a tertiary care teaching hospital in a rural region of central India.Patient selectionThe inclusion criteria were patients aged 18 years and above with a diagnosed case of SCH as per the American Thyroid Association guidelines [MethodsA serum sample was collected in a fasting state (12-hour fasting state) with a sterile venipuncture technique. The sample was then processed for lipid profile, thyroid profile , and anti-TPO antibody. Lipid estimations were done using an automated analyzer and commercial kits. We adhered to the National Cholesterol Education Program Adult Treatment Panel III guidelines for serum lipids. According to these guidelines, hypercholesterolemia is defined as a TC value ≥200 mg/dl, high LDL as an LDL level ≥130 mg/dl, hypertriglyceridemia as a TG value ≥150 mg/dl, and low high-density lipoprotein (HDL) as an HDL value <40 mg/dl [Study sample sizeThe sample size of this case-control study comprised a total of 150 individuals. A total of 75 patients diagnosed with SCH were included in the case group and 75 healthy matched individuals were recruited as the control group.Data entry and analysisStatistical analysis was performed using SPSS Statistics version 23 . Continuous variables were presented as mean ± standard deviation (SD) and categorical variables were presented as absolute numbers and percentages. Differences between groups were assessed with the chi-squared or Fisher's exact test for categorical variables as appropriate. The association of anti-TPO antibodies with various components of lipid level was assessed using multiple regressions. A p-value <0.05 was considered statistically significant.Ethical considerationsThe study was conducted after obtaining approval from the Ethics Committee of Uttar Pradesh University of Medical Sciences.Confidentiality of dataStrict confidentiality was maintained regarding the information obtained about the patients, and the identity of the patients was not revealed. Data, if shared, were anonymized.The study consisted of 150 patients (75 cases and 75 controls) satisfying the inclusion and exclusion criteria; 77.3% of the subjects were female and 22.7% were male. The study showed anti-TPO positivity among 82.7% of cases of SCH as depicted in Table This study also demonstrated the association between lipid profile and anti-TPO antibody as depicted in Table Our study showed that serum HDL was low in 15.4% of cases in the negative anti-TPO antibody SCH sub-group and 24.2% of cases in the positive anti-TPO antibody SCH sub-group (p=0.490). Serum VLDL was elevated in 84.6% of cases in the negative anti-TPO antibody SCH sub-group and 83.9% of cases in the positive anti-TPO antibody SCH sub-group (p=0.947). Additionally, 82.8% of participants with anti-TPO antibodies within the normal range had normal LDL levels, while 11.1% of participants with elevated anti-TPO antibodies had normal LDL levels (p<0.001) as depicted in Table The study showed a correlation coefficient of 0.0432 between serum TG and anti-TPO antibody level as shown in Figure SCH is defined as a state of elevated TSH level with a normal level of thyroid hormones. Various epidemiological studies in India have shown a prevalence rate of 9-11.4% for SCH among the general population . SCH is In their study of 50 cases, Jayashankar et al. found anti-TPO positivity among 50% of SCH cases , whereasSrivastava et al. in their study found that dyslipidemia was present in 100% of SCH patients with positive anti-TPO antibodies . In the The current study, while exploring the association of lipid profile with anti-TPO antibody, found that SCH with anti-TPO antibody positivity is significantly associated with raised serum cholesterol as well as raised serum TG and serum LDL levels as depicted in Figure In the Rotterdam study, SCH was present in 10.8% of participants and was associated with a greater age-adjusted prevalence of aortic atherosclerosis  and myocardial infarction  . It was To summarize the findings, our study has found a high prevalence of anti-TPO positivity in SCH. Additionally, we have found a significant association of dyslipidemia with anti-TPO positivity. The association between dyslipidemia and SCH is of great significance as dyslipidemia is a well-known risk factor for hypertension, cardiovascular disease, and stroke. The current study further highlights the importance of routine monitoring of lipid profiles in cases of SCH to timely identify any derangements to prevent adverse outcomes. We strongly recommend further investigation on the use of thyroxine in patients with SCH for dyslipidemia.To the best of our knowledge, this is the first study conducted on this topic in a rural region of central India; however as this study was conducted at a single institute, further studies are required to validate our findings before we can extrapolate them to the general population. Secondly, as the nature of our study design was case-control, this study can be used to establish a correlation but cannot establish causation. Hence, a large-scale prospective study is required to confirm the association between dyslipidemia and SCH.This study showed an increased incidence of dyslipidemia in patients with elevated anti-TPO antibodies. SCH with positive anti-TPO antibody is significantly associated with elevated serum TC, serum TG, and serum LDL levels. Dyslipidemia is a significant risk factor for cardiovascular disease and mortality. Hence, early screening and timely diagnosis of dyslipidemia are recommended to prevent cardiovascular morbidity and mortality in SCH patients with positive anti-TPO antibodies. Further, we recommend exploring the role of thyroxine as an intervention to target the problem of dyslipidemia in patients with SCH, particularly in anti-TPO antibody-positive patients.


Results: Patients showed a significant improvement of WOMAC total score, subscale pain, subscale stiffness and subscale function from preoperative to the follow up after 12 months in both groups, with significantly superior results for the WOMAC total score for the ERAS group (p = 0.042). EQ-5D and EQ-5D VAS showed a significant improvement from preoperative to 12 months postoperative (p < 0.001) for both groups, while no difference regarding the group-comparison was shown. Conclusion: Health-related quality of life and functional outcome increased to excellent values after total hip arthroplasty with the use of an enhanced recovery concept and a conventional rehabilitation, with a superior WOMAC total score for ERAS and a tendency to better results for health-related quality of life for patients with ERAS within the follow up after 12 months.Background: The concept of enhanced recovery after total hip arthroplasty is gaining worldwide interest, as it shortens the length of hospital stay without an increase of complications. The aim of the study was to investigate the functional outcome and health-related quality of life 12 months after cementless total hip arthroplasty with the use of an enhanced recovery concept in comparison to a conventional rehabilitation. Material and Methods: 320 patients were retrospectively analyzed who underwent primary cementless total hip arthroplasty (THA). A total of 123 of the patients received an enhanced recovery program (ERAS) and 197 patients a conventional rehabilitation (Non-ERAS). Twelve months postoperatively, a clinical examination was performed regarding satisfaction, function and pain. Results were evaluated using WOMAC, EQ-5D-5L and EQ-VAS. A 1:1 matching was performed to correct for confounding variables, regarding age, sex and ASA score. Finally, 122 patients ( The rising prevalence of osteoarthritis has led to an increase in primary total hip arthroplasty (THA) over the past two decades . Since 2THA has shown to be an effective operation for symptomatic osteoarthritis. Despite its efficacy, 7–15% of patients still express dissatisfaction after the procedure . The reaThe aim of the study was to evaluate the functional outcome and health-related quality of life after a primary cementless total hip arthroplasty with an enhanced recovery concept in comparison to a conventional rehabilitation. We hypothesized that patients with an enhanced recovery protocol would show a better functional outcome and higher postoperative satisfaction.® femoral stem, DePuy Pinnacle ® acetabular component) between mid 2018 and mid 2019 in a single center. Inclusion criteria were primary THA using a DePuy Corail® femoral stem due to primary or secondary osteoarthritis and a complete dataset of the collected scores. In the chosen period between 2018 and 2019, the ERAS setup was established parallelly in a part of the unit, and therefore, both setups could be ideally compared. Since both concepts were offered at the same time, the patient was able to choose one independently after knowing the differences between the treatment concepts. For all patients, an anterolateral approach was used. Exclusion criteria were revision surgery, severe congenital disorder of the hip, obesity III° (BMI > 40 kg/m2), malignancy and immobility. The data used were taken from digitized patients records. The local ethics committee granted ethical approval (reference number 19-1352-104). The study was applied in accordance with the ethical standards of the Declaration of Helsinki 1975.In this retrospective study, 320 patients were included who underwent primary cementless, collarless THA  was administered. As an anesthetic technique, a short-acting spinal anesthesia was used (prilocaine 1% hyperbaric 4 mL with sufentanil 10 µg and dexamethasone 8 mg i.v. as the standard). Furthermore, tranexamic acid was administered topically (2 g) and intravenously (1 g). A local-infiltration analgesia was applied in the periacetabular and femoral region, as well as subcutaneously . In general, drains were not used. Postoperatively, full weight-bearing of the operated extremity was permitted. The first mobilization of the patient took place 2–3 h after surgery. Physiotherapeutic treatment was performed twice a day during the hospital stay and included exercises for muscle strengthening, tutorials to improve coordination and gait training under hip precautions. The physiotherapists were specially educated for the concept of ERAS. A treatment protocol for THA with the concept of ERAS was established in our department.A total of 197 of 320 patients received a conventional recovery program after THA (non-ERAS group). As an anesthetic technique, a long-acting spinal anesthesia was used (bupivacain 0.5% = 4 mL). In contrast to the ERAS group, no NSAID was administered preoperatively. No tranexamic acid or local anesthesia was applicated during surgery. In general, wound drains were used. Patients were instructed to walk with crutches with full weight-bearing, according to the traditional postoperative recommendations of the unit. The first physiotherapeutic treatment was performed on the first postoperative day and took place once a day in consideration of hip precautions.For optimizing postoperative pain, a pain management concept was established regarding the recommendations within the WHO analgesic ladder . For theIn our department, all THA patients were encouraged to complete several scores like the EQ-5D-5L, EQ-5D VAS and WOMAC. Euroqol-5D-5L has five dimensions of health status, with mobility, self-care, usual activities, pain/discomfort and anxiety/depression. Depending on the level, a digit is assigned to each dimension, so that you get a five-digit number combination . Euroqol VAS (EQ VAS) has a scale of 0–100 points. A score of 0 points represents the worst possible health status, while 100 points represents the best possible health status.The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score includes 24 questions with respect to three subscales, i.e., pain, stiffness and physical function. The WOMAC Likert version was used, with a scale from 0 to 4 points for each question. The ranges of scores for the respective subscales are as follows: pain 0–20 points, stiffness 0–8 points and physical function 0–68 points, with the higher the value, the worse the result. All cases were reevaluated 12 months postoperatively, including a physical examination and evaluation of EQ-5D-5L, EQ-VAS and WOMAC.n = 61, in each group, The groups of ERAS and non-ERAS were adjusted in accordance with the matched pair method to get comparable groups in size and a distribution of confounders. The 1:1 matching was performed according to age, sex and ASA score. If there was more than one matching partner for one patient, one patient was randomly chosen. Thus, a total of 122 patients .Normally distributed data were represented by mean values and standard deviation. The median  was used for non-normally distributed continuous or ordinal variables, while absolute and relative frequencies were used for categorial variables. A comparison of the scores from preoperative to postoperative were performed by using Wilcoxon signed rank test. Differences between the two groups regarding the quality of life measurements were assessed by using the Mann–Whitney-U-test. No adjustments of the significance level for multiple comparisons were done. The significance level was set to n = 61 per group, respectively) could finally be statistically analyzed (p = 0.042 (p < 0.001)  .p = 0.889). The median EQ-5D preoperatively was 0.62  for the non-ERAS group and 0.66  for the ERAS group. Twelve months postoperatively, the ERAS and the Non-ERAS group improved for EQ-5D to 1.00 , respectively, p < 0.001, respectively), The median Euroqol 5D-5L (EQ-5D) preoperatively showed no significant difference between the two groups (p = 0.183). The non-ERAS group improved 12 months postoperatively for EQ-5D VAS to 85.00 , and the ERAS group improved to 90.0 . An overview of the values of EQ-5D VAS is shown in p < 0.001, respectively).The median EQ-5D VAS preoperatively was 40.00  for the non-ERAS group and for the ERAS group 50.00 . The EQ-5D VAS preoperatively was statistically not significant between the two groups  for both groups, ERAS and Non-ERAS. In the follow up after 12 months, both groups had a median EQ-5D of 1.0 for the ERAS and non-ERAS groups. The studies of Larsen et al. [The health-related quality of life (HRQoL) was measured by Euroqol (EQ-5D). From preoperative to the follow up after 12 months, the improvement of EQ-5D was statistically significant  and relatively fit (predominantly ASA was 2). Furthermore, the ERAS concept was newly established in our department, which could have caused a selection bias. As the maximum follow up in this study was limited to 12 months, long-term results are not available. Another point which could have influenced the results is the difference of inpatient rehabilitation or outpatient rehabilitation. There was no direct comparison between the two rehabilitations. A randomized controlled study design or further prospective long-term studies should be undertaken to measure and quantify health-related quality of life and functional outcome after total hip arthroplasty with the concept of enhanced recovery.Total hip arthroplasty with the use of an enhanced recovery concept and a conventional rehabilitation showed a high patient satisfaction in the present study. In a follow-up period of 12 months, pain scores decreased significantly, and HRQoL scores increased to excellent values, and even higher values than the population norm were achieved. We could show that an ERAS concept tends to lead to a better outcome after THA compared to a conventional concept, with statistically significant superior functional scores  in the matched study population.


Nasopharyngeal carcinoma (NPC) is an aggressive tumor with a complex etiology. Although Epstein–Barr virus (EBV) infection is known environmental factor for NPC development, the degree to which EBV naturally infects nasopharyngeal epithelium and the moment when and why the virus actively begins to affect cell transformation remains questionable. The aim of this study was to explore the association between LMP1 gene variability and potential contribution to NPC development. A systematic review was performed through searches of PubMed, Web of Science (WoS) and SCOPUS electronic databases. Additionally, meta-analysis of the difference in the frequency of seven LMP1 gene variants in NPC and control individuals was accomplished. The results from this study give a proof of concept for the association between 30 bp deletion  and Xhol loss  and NPC susceptibility when comparing biopsies from NPC and healthy individuals. Otherwise, 30 bp deletion from NPC biopsies could not distinguish NPC from EBV-associated non-NPC tumors . However, B95-8, China1 and North Carolina variants were uncommon for NPC individuals. Much more efforts remains to be done to verify the biological significance of the differences observed, define so-called “high-risk” EBV variants and make it available for clinical application. Nasopharyngeal carcinoma (NPC) is a rare but aggressive tumor that originates from the epithelial cells of the retronasal cavity. Although it could be presented with varying degrees of differentiation, the undifferentiated carcinoma of nasopharyngeal type  is the most dominant histopathological type in high-risk areas. Unlike most of the world’s population, including Europe and USA where NPC is rare with an incidence below 1 per 100,000 persons per year, in endemic regions of Asia incidence rate is 20–30 per 100,000 persons per year . On the A20 and bcl-2 genes, a modulation of the morphology and motility of epithelial cells, a downregulation of metastasis suppressors, promotion of angiogenesis and activation of proinflammatory cytokines [The association of NPC and EBV was first discovered by seroepidemiological studies which revealed elevated anti-EBV IgA antibodies in patients with NPC ,7. In adytokines . It has ytokines .LMP1 is a 356 amino acid integral membrane protein formed by three domains: a short N-terminal tail (amino acids 1–23); six transmembrane domains (amino acids 24–186); the long cytoplasmic C-terminal tail (amino acids 187–386) with three distinct functional domains or the C-terminal activation regions (CTAR) 1, 2, and 3 ,15. InveUnderstanding EBV molecular epidemiology could be crucial in preventing and treating all pathological conditions associated with viral infection. Considering the heterogeneity of the examined populations in previous studies, there are the ambiguous results and the lack of definitive conclusions about the association between LMP1 gene variability and potential contribution to NPC development. The aim of this systematic review and meta-analysis was to explore this problem.n = 1619) removed, title and abstracts were evaluated for 1801 articles. In total, 1747 articles were excluded because they were not original articles, did not explore NPCs, did not compare NPC and control groups, examined populations other than human , explored genes other than LMP1, explored the presence of LMP1 and/or its expression level but not LMP1 variants, or were abstracts. Of the 54 reviewed full text articles, 31 were selected for inclusion in the systematic review. A flow diagram illustrating this selection process is presented in A total of 3420 potentially eligible articles were found. After duplicates . Other sources for EBV LMP1 variants detection from NPC patients were peripheral blood in 8 studies , throat washing in 5, and xenografts in 1 study. Control groups were very heterogeneous. Most common sources of a control sample were patients with lymphomas, then non-NPC tumors, infectious mononucleosis, nasopharyngeal inflammation, and oral hairy leukoplakia. Three ways of sampling were implemented: biopsy, blood sampling and throat washing.The age of the participants ranged from 2 to 86 years; however, the predominant age was approximately 50 years. In the NPC group age ranged from 2 to 86 years and in the control group from 5 to 77 years. Regarding the gender of the participants, there was a male predominance with a male/female ratio of 1.87 .Summary of findings from this study is presented in the Meta-analysis was performed for 7 variants . First, it was accomplished regardless of the country of patient origin, and then by regions (endemic and non-endemic). Only studies that evaluated and compared the frequency of LMP1 gene variants in human NPC and control groups were taken into account.p < 0.001) (p < 0.001) (p < 0.001)  , and eve< 0.001) . The pre< 0.001) .A total of 27 studies were included in the meta-analysis of the association between 30 bp deletion LMP1 and NPC susceptibility. Due to the great tissue heterogeneity, the meta-analysis was organized in subgroups according to the type of clinical samples. First, this relationship was evaluated between NPC biopsy samples and other specimens . Further, it was examined in non-biopsy samples (throat washings and blood) between NPC and healthy controls.p = 0.004) (p < 0.001) (p = 0.160)   . The res< 0.001) . However= 0.160) . In addictively) .p = 0.290, and OR = 2.22, 95% CI = 0.26–18.60, p = 0.460)  .p < 0.001)   . In addictively) .p = 0.003) (p = 0.150) (p = 0.430)  . Otherwi= 0.150) , as well= 0.430) .p = 0.760) (p = 0.500) (p = 0.220)  . Additio= 0.500) . Med LMP= 0.220) .p = 0.040)  .p = 0.030) (p < 0.001) (p = 0.170)  , but als< 0.001) . Otherwi= 0.170) .Additionally, there was no association between 30 bp del and NPC susceptibility in studies from non-endemic regions when comparing biopsies from NPC and EBV-associated non-NPC tumors patients  .p = 0.003) (p = 0.070)  , but the= 0.070) .This study provided a comprehensive systematic review of LMP1 gene variability, not only between NPC and non-NPC participants in general, but a much more homogeneous and detailed comparison. For the first time, a comparative analysis of LMP1 gene variability included seven variants: Xhol loss, 30 bp and 69 bp deletions, B95-8, China1, Mediterranean, and North Carolina variants, which were presented in different tumor-altered and healthy tissues. Finally, a comparison of the frequency of LMP1 gene variants was also made in relation to geographical origin of the clinical sample.The course of the NPC pathogenesis is influenced by several factors, among which are genetic susceptibility, environmental and viral factors. As far as viral factors are concerned, EBV and Human papilloma virus (HPV) infections are the most investigated and mentioned in this context. Only recently, the results of meta-analysis designed to establish the relationship between viral combinations in NPC stratified according to histological type have been published . While kSeveral different viral oncogenes such as LMP1, LMP2, and EBNA1 play an important role in the pathogenesis of EBV-associated tumors. The majority of the studies, concerning EBV genetic variability, have focused attention and investigation on LMP1 gene polymorphisms, which is at the same time the most important EBV oncogene but also the most variable gene. Having this in mind, a previous meta-analysis determined the impact of 30 bp deletion and Xhol loss on the risk for NPC development . The autThe systematic classification of the clinical samples’ origin and their comparative analysis improved the validity of results obtained in this study. Therefore, it becomes clearer that the specificities of the viral genome are primarily related to tumor-altered tissue, not only NPC but also tissues of the other EBV-related tumors which were included and analyzed in this meta-analysis. Thus, from our results, it could be seen that the association between the LMP1 deleted variant and NPC was seen in viral genome which was localized in the tumor-altered tissue itself, and that it is absent when the frequency of 30 bp deletion is compared with any other type of clinical sample. These findings support published data about the time specific and determined role of EBV oncogenic activity in early phases of NPC development ,56. ActuExtensive research of LMP1 gene heterogeneity has so far defined more than seven different strains. The functional differences between the LMP1 of epithelial cancer-derived EBV, which belongs to China1, and lymphoid-derived EBV, which belongs to B95-8, are well described . HoweverMeta-analysis by regions reveals that the presence of 30 bp deletion in the NPC biopsies was almost seven times higher than in healthy tissue in endemic regions. As it has long been postulated that distinct EBV strains are predominant in areas with a high incidence of the NPC, and that EBV strain variation contributes to the endemic nature of the NPC, 30 bp deletion and Xhol loss in these areas could serve as prognostic markers for tumor development ,62. ThisAs the EBV genetic variability led to the increasing interest of possible disease-specific associations with EBV variants, but still too diverse to single out the individual risk factors, new high-throughput sequencing technologies marked a new era of EBV sequencing. Therefore, a recent explosive increase in EBV whole genome sequences could open the window of new opportunities in EBV genome research, and lead to identification of high-risk groups of people who are predisposed to the NPC and impact on the early diagnosis of the NPC . In addiThis is not the first, but it is a comprehensive meta-analysis of the association between LMP1 variants and the NPC susceptibility. Regardless of its advantages, some limitations were recognized. First, only articles in English were included (there were 67 articles in other languages). Second, abstracts were not taken into account. The first and the second reason might lead to making a selection bias and losing the data. In order to examine publication bias, Funnel plots were performed, and they indicated no publication bias. Third, there was great heterogeneity in types of clinical samples, so it was chosen to do meta-analysis in subgroups, according to the types of clinical samples taken from NPC and control groups to make more homogeneous conclusions. Fourth, the number of studies in each meta-analysis was different. Some of them, with smaller number of included studies (three) had smaller statistical power than those with greater number of included articles. Fifth, because of the scarcity of data with potential influence on the association between LMP1 variants and NPC susceptibility, such as age and gender, we were not able to perform the meta-regression analysis in order to control the estimated effect size (OR) and avoid underestimation and overestimation in these situations. Sixth, regardless of the duration of performing studies on the association between LMP1 variants and NPC (from 1973 to today) there was not enough survival data in order to perform meta-analysis of hazards.In summary, the results from this study give a proof of concept for the association between two LMP1 variants (30 bp deletion and Xhol loss) and the NPC susceptibility when comparing biopsies from the NPC and healthy individuals. Moreover, this association was also found with other EBV-associated tumors. Otherwise, 30 bp deletion from NPC biopsies could not distinguish NPC from EBV-associated non-NPC tumors. On the other hand, B95-8, China1 and North Carolina variants were uncommon for NPC individuals. Although it is clear that not only EBV LMP1 gene variability but also many other non-viral factors are involved in the etiopathogenesis of the NPC, the importance of identification of so-called “high-risk“ EBV variants could be crucial in defining relative variant tropism, designing targeted antiviral candidates or even make progress toward an EBV vaccine. Over the past decades, numerous efforts have been made, but much more work remains to be done to verify the biological significance of the differences observed and make it available for clinical application.https://osf.io/znrwj/) [This systematic review was performed in accordance with the PRISMA protocol  and MOOSE guidelines for observational studies after systematic review registration at PROSPERO (Systematic review registration statement is available at /znrwj/) ,65.Publications were screened for inclusion in the systematic review in two phases, and all disagreements were resolved by discussion at each stage with inclusion of a third reviewer. We included studies that detected LMP1 gene variants in patients with nasopharyngeal carcinoma and any other group for comparison. Studies were eligible for inclusion if LMP1 gene variants were detected in both groups. Studies were excluded if they (i) investigated other outcomes, (ii) did not make comparisons between patients with nasopharyngeal carcinoma and control groups, (iii) examined other populations , (iv) assessed other genes , (v) did not assess LMP1 variants, but its function or expression, (v) were abstracts, or (vi) were not original articles.Two biostatisticians with expertise in conducting systematic reviews and meta-analyses  developed the search strategy. A systematic review of peer-reviewed publications was performed through searches of PubMed, Web of Science (WoS) and SCOPUS electronic databases until 28 April 2021. Search queries differed according to the database. Keywords for the PubMed search were  and  and (TNF receptor associated factor 2 or LMP1-Associated Protein 1 or LMP1 or variant or mutation or deletion or polymorphism); for WoS: ALL =  AND ALL = (Epstein Barr Virus OR Epstein-Barr Virus OR EBV OR Burkitt's lymphoma virus OR Human herpesvirus 4 OR HHV4) AND ALL = (TNF Recept associated factor 2 OR LMP1-Associated Protein 1 OR LMP1 or variant or mutation or deletion or polymorphism), and for SCOPUS:  OR TITLE-ABS-KEY  AND TITLE-ABS-KEY (“Epstein Barr Virus”) OR TITLE-ABS-KEY (“Epstein-Barr Virus”) OR TITLE-ABS-KEY (“EBV”) OR TITLE-ABS-KEY (“BURKITT’S LYMPHOMA VIRUS”) OR TITLE-ABS-KEY (“Human herpesvirus 4”) OR TITLE-ABS-KEY (“HHV4”) AND TITLE-ABS-KEY (“TNF RECEPT ASSOCIATED FACTOR 2”) OR TITLE-ABS-KEY (“LMP1-Associated Protein 1”) OR TITLE-ABS-KEY (“LMP1”) OR TITLE-ABS-KEY (“variant”) OR TITLE-ABS-KEY (“mutation”) OR TITLE-ABS-KEY (“deletion”) OR TITLE-ABS-KEY (“polymorphism”). Only publications in English were taken into account. In addition, reference lists of articles identified through electronic retrieval were manually searched, as well as relevant reviews and editorials. Experts in the field were contacted to identify other potentially relevant articles. Authors of relevant articles were contacted to obtain missing data.Two reviewers  independently evaluated the eligibility of all titles and abstracts. Studies were included in the full text screening if either reviewer identified the study as being potentially eligible, or if the abstract and title did not include sufficient information. Studies were eligible for full text screening if they included detection of LMP1 gene variants in patients with nasopharyngeal carcinoma and control group. The same reviewers independently performed full text screening to select articles for inclusion according to the criteria listed under Inclusion and Exclusion Criteria. Disagreements were resolved by consensus  or arbitration .https://osf.io/znrwj/.Two reviewers independently abstracted the following data: author(s), country of research, year of publication, study design, sample size, study population, EBV positivity, type of LMP1 gene variant, method for detection of LMP1 gene variant. Each reviewer independently evaluated the quality of selected manuscripts using an adapted version of the Newcastle–Ottawa tool for observational studies . Reviewehttps://osf.io/znrwj/.). A p value <0.05 was considered to be statistically significant. Analyses were performed using Review Manager Version 5.4 [The primary outcome was the frequency of LMP1 gene variants. As the outcome is dichotomous and the sample size varies Mantel–Haenszel method was used as a measure of effect size to examine the differences in ratio of a specific LMP1 gene variant in evaluated study groups from all primary articles. Mantel–Haenszel method is a fixed-effect meta-analysis method that uses a different weighting scheme that depends on which effect measure is being used. Heterogeneity was assessed using the Chi-square Q test and I2 statistic. I2 presents the inconsistency between the study results and quantifies the proportion of observed dispersion that is real, i.e., due to between-study differences and not due to random error. The categorization of heterogeneity was based on the Cochrane Handbook  and statsion 5.4 .


Trypanosoma cruzi (T. cruzi) is a hemoflagellate protozoan parasite that causes Chagas disease, a neglected tropical disease that affects more than 6 million people around the world, mostly in Latin America. Despite intensive research, there is no vaccine available; therefore, new approaches are needed to further improve vaccine efficacy. It is well established that experimental T. cruzi infection induces a marked immunosuppressed state, which includes notably increases of CD11b+ GR-1+ myeloid-derived suppressor cells (MDSCs) in the spleen, liver and heart of infected mice. We previously showed that a trans-sialidase based vaccine (TSf-ISPA) is able to confer protection against a virulent T. cruzi strain, stimulating the effector immune response and decreasing CD11b+ GR-1+ splenocytes significantly. Here, we show that even in the immunological context elicited by the TSf-ISPA vaccine, the remaining MDSCs are still able to influence several immune populations. Depletion of MDSCs with 5 fluorouracil (5FU) at day 15 post-infection notably reshaped the immune response, as evidenced by flow cytometry of spleen cells obtained from mice after 21 days post-infection. After infection, TSf-ISPA-vaccinated and 5FU-treated mice showed a marked increase of the CD8 response, which included an increased expression of CD107a and CD44 markers in CD8+ cultured splenocytes. In addition, vaccinated and MDSC depleted mice showed an increase in the percentage and number of CD4+ Foxp3+ regulatory T cells (Tregs) as well as in the expression of Foxp3+ in CD4+ splenocytes. Furthermore, depletion of MDSCs also caused changes in the percentage and number of CD11chigh CD8α+ dendritic cells as well as in activation/maturation markers such as CD80, CD40 and MHCII. Thus, the obtained results suggest that MDSCs not only play a role suppressing the effector response during T. cruzi infection, but also strongly modulate the immune response in vaccinated mice, even when the vaccine formulation has a significant protective capacity. Although MDSC depletion at day 15 post-infection did not ameliorated survival or parasitemia levels, depletion of MDSCs during the first week of infection caused a beneficial trend in parasitemia and mice survival of vaccinated mice, supporting the possibility to target MDSCs from different approaches to enhance vaccine efficacy. Finally, since we previously showed that TSf-ISPA immunization causes a slight but significant increase of CD11b+ GR-1+ splenocytes, here we also targeted those cells at the stage of immunization, prior to T. cruzi challenge. Notably, 5FU administration before each dose of TSf-ISPA vaccine was able to significantly ameliorate survival and decrease parasitemia levels of TSf-ISPA-vaccinated and infected mice. Overall, this work supports that targeting MDSCs may be a valuable tool during vaccine design against T. cruzi, and likely for other pathologies that are characterized by the subversion of the immune system. Trypanosoma cruzi (T. cruzi) is the etiological agent of Chagas disease. More than 6 million people are infected and 65-100 million people live in areas at risk of T. cruzi infection worldwide , and M-MDSCs   . Severalc-MDSCs) .T. cruzi infection. For instance, the percentage of CD11b+ GR-1+ (Ly6G+/Ly6C+) cells in total spleen cells may increase from 2-5% to nearly 20-30% in acutely infected mice  was able to decrease spleen MDSCs (mainly G-MDSCs) while the number of Tregs in the spleen was increased, a result that correlated with a significant increase of mouse survival against T. cruzi infection  used in all experimental procedures were obtained from the Centro de Medicina Comparada, Instituto de Ciencias Veterinarias del Litoral (ICIVET-CONICET) Universidad Nacional del Litoral (UNL), Argentina. All protocols for animal studies were approved by the Animal Care & Use Committee of the Facultad de Bioquímica y Ciencias Biológicas, UNL, according to the Institutional guidelines that are based on international ethical guidelines for biomedical research involving animals.BALB/c mice (n =5-6/group) were immunized with three subcutaneous doses, one every two weeks, containing 10 μg of a fraction of the trans-sialidase protein (TSf)  with 3 uControl groups were immunized with phosphate buffered saline (PBS) solution, following the same protocol. For infection, mice were challenged intraperitoneally with 900 or 1500 bloodstream trypomastigotes of Tulahuen strain, as indicated, 15 days after the last immunization. Parasites employed belong to the discrete typing unit VI (DTU VI) and were maintained by serial passages in Cbi suckling mice. Cbi mice were bred in the animal facilities of School of Medicine of Rosario, following protocols for animal studies approved by the Institutional Bioethics and Biosecurity Committees (Resolution N 3913/2008). According to the experiment, mice were treated intraperitoneally with 5FU (Sigma Aldrich) 50mg/kg, a dose previously described in a similar model of infection . 5FU treT. cruzi with the dose employed  in treated mice . We alsoemployed .Parasitemia was monitored at day 20 p.i. by examining 5 μl of blood directly under microscope (Leica Microsystems), as previously described . Surviva4Cl. Then, splenocytes were re-suspended in PBS with 3% fetal bovine serum (Gibco) and 0,1% azide for direct flow cytometry, or in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum, 2% penicillin (100 μg/mL) and streptomycin (100 U/mL) (Gibco) and 0.4 mM 2-mercaptoethanol for in vitro culture.Groups of mice were immunized with TSf-ISPA or PBS and treated or not with 5FU as described above. At day 21 p.i., mice were sacrificed and spleens were aseptically harvested and homogenized. Absolute numbers of spleen were determined by counting cells in Neubauer chamber using Turk solution and then red blood cells were eliminated by lysis with a solution of NH6 cells were first incubated with anti-FcγIII/II receptor antibody and then stained with appropriate combinations of the following antibodies from BD-Pharmingen: anti-CD11b, anti-Ly6G, anti-Ly6C, anti-CD4, anti-CD11c, anti-CD80, anti-CD40, anti-MHCII, and anti-CD8α according to the manufacturer´s instruction in appropriate combinations of fluorophores for posterior analysis. The MFI index of CD40 in CD11chigh dendritic cells was calculated by dividing the MFI of CD40 in each group of mice/MFI of CD40 in non-infected mice. A similar formula was used to calculate the MFI index for CD80 and MHCII.For flow cytometry, 1×10Intranuclear staining with anti-Foxp3 from Miltenyi Biotec was used following the manufacturer´s instruction.in vitro culture, 1×106 cell/mL/well were cultured in 24 or 48-well plates (Nunc) in supplemented RPMI. Splenocytes were stimulated with concanavaline A (ConA) (Sigma-Aldrich) (5ug/ml) and T. cruzi homogenate or RPMI as control. After 72 hours at 37°C and 5% CO2, cells were harvested, incubated with anti-FcγIII/II receptor antibody and then stained with anti-CD8α, anti-CD107a and anti-CD44 in appropriate combinations of fluorophores for posterior analysis  and analyses were performed using FlowJo software.6 cell/mL/well) from each experimental group  were cultured in 24 or 48-well plates (Nunc) in supplemented RPMI. Cells were stimulated with ConA (5ug/ml) and T. cruzi homogenate or RPMI as control. In addition, aminoguanidine 0.5 mM, a well-known iNOS inhibitor, was added to some wells as indicated in the Figures. After 72 hours 0.5 mg/ml MTT  was added to each well. After 4 additional hours, culture media were removed and 100 ul DMSO was added to each well. Finally, optical density (OD) was measured as previously reported . Significance is indicated with (*) when p<0.05 between the indicated groups.T. cruzi of Tulahuen strain. Notably, similar results have been obtained concerning the increase in parasitemia and mortality from day 15 to day 21 p.i.  was used to selectively deplete MDSCs, as previously shown in several models, including models of T. cruzi infection  succumbed to the parasite challenge before day 21 p.i., likely as a consequence of an exacerbated immune response, as previously suggested  increased in a manner that correlated with the spleen size of the groups analyzed: NI<=TSf-ISPA Tc+ 5FU<TSf-ISPA Tc+< PBS Tc+ mice <TSf-ISPA Tc+ 5FU mice<TSf-ISPA Tc+ mice< PBS Tc+ mice , a selective iNOS inhibitor. As expected, PBS Tc+ mice showed a notable increase in the percentage and absolute number of spleen G-MDSCs cells expressing iNOS (CD11b+ Ly6G+ iNOS+) as compared to non-infected mice. In addition, TSf-ISPA Tc+ mice showed lower levels of spleen G-MDSCs expressing iNOS than PBS Tc+ mice, a result that correlates with the decrease of G-MDSCs observed in TSf-ISPA mice, as previously reported . FinallyT. cruzi infected mice express iNOS, which is a feature of MDSC cells and has been involved as an important mechanism of suppression by these cells during acute T. cruzi infection. In addition, the depletion of MDSCs almost overlapped with iNOS+ expressing cells reduction, strongly suggesting that MDSCs largely accounts for the increase of iNOS+ splenocytes after T. cruzi infection. Moreover, the addition of AG to splenocytes cultured with ConA increased proliferation, which strongly supports the immunosuppressor role of MDSC iNOS+ cells in our experimental condition. Additionally, splenocytes from TSf-ISPA Tc+ and TSf-ISPA Tc+ 5FU mice,  showed higher expression of Ki-67 in CD4+ cells than splenocytes from PBS Tc+ mice, after in vitro culture with ConA  showed a marked increase in Foxp3+ expression in CD4+ cells and in the percentage of Tregs, compared to PBS Tc+ and TSf-ISPA Tc+ mice , an important percentage of survival, of between 80-100% was obtained for the TSf-ISPA Tc+ 5FU-treated (pre-vaccine) mice , as well as vaccinated and MDSC depleted mice, showed a significant increase in some of those activation markers, which correlated with protection capacity and the elicitation of an increased effector response.It has been described that response . Moreoveresponse . In our T. cruzi infection is the dual role attributed to several components of the immune system. For instance, it has been reported that DCs are a double-edged sword , a vaccine should be able to decrease the signal 1 of growth factors to avoid an increase of MDSCs. This hypothesis and the “two-signal” model were supported by the finding that TSf-ISPA-vaccinated group of mice has lower levels of plasma GM-CSF, which correlated with lower levels of MDSCs, allowing an increase in the levels of plasma IFN-γ.An interesting point considering ed sword , and in ed sword . Tregs csistence . Reactivsistence . INF-γ infection . Thus, infection , an incrT. cruzi than vaccinated mice that did not receive 5FU, supporting that MDSCs could be targeted in a beneficial manner to enhance the protective capacity of a vaccine candidate.Taken together, the studies performed by depleting MDSCs at day 15 p.i. suggest that those cells are strongly beneficial for parasite persistence, allowing the survival of its host and widely influencing the immune response, even when the mice were previously immunized with a vaccine with a significant protective capacity. The fact that MDSC depletion at day 15 p.i. increased the CD8 effector response but did not cause an improvement in the survival of vaccinated and infected mice, suggests that other strategies would be needed to support the targeting of MDSCs as a valuable tool to enhance the protective capacity of a vaccine candidate. For this purpose, TSf-ISPA vaccinated mice were early treated with 5FU one day before infection and at day 5 p.i. TSf-ISPA-vaccinated mice that were treated with 5FU at the beginning of infection showed higher survival against a high dose of T. cruzi, improving the survival of TSf-ISPA-vaccinated mice that were not treated with 5FU. In line with these results, parasitemia levels were also decreased in TSf-ISPA mice that were pretreated with 5FU as compared to immunized mice that did not received 5FU. Although survival is likely the most clear and relevant parameter of protection, parasitemia levels may also have important implications in the context of T. cruzi infection. In this sense, a vaccine that decreases parasite load could be useful to avoid the development of chronic Chagas disease and to reduce potential vertical transmission  and the regulatory arm of the immune system (Tregs); b) although MDSC depletion at day 15 of infection supports that those cells are absolutely necessary for mouse survival, benefiting the parasite, the present results suggest that in the context of vaccination, MDSC could be removed during early infection improving the host survival; c) 5FU treatment at the stage of immunization enhanced the protective capacity of the TSf-ISPA vaccine, supporting the possibility to use this strategy in rational vaccine design not only against T. cruzi but also in other pathologies that are characterized by the subversion of the immune system.Overall, throughout this study we have shown that: a) MDSCs have a wide The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Animal Care & Use Committee of the Facultad de Bioquímica y Ciencias Biológicas, UNL.T. cruzi strain used in all the experiments. AP, IM, and GC analyzed the data, designed the figures, and wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.GC and IM designed the experiments. JG and CR performed most of the experiments. EP and IB optimized the protocols and obtained the TSf protein. In addition, they assisted in some experiments. GL produced the ISPA adjuvant and assisted in the experiments. CP and MV designed the experiments and analyzed the data of dendritic cells. AP produced the Tulahuén This work was supported by ANPCyT  , CONICET  and the Universidad Nacional del Litoral, Argentina.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Major depressive disorder (MDD) is one of the most common psychiatric disorders and is a great disease burden. However, its underlying pathophysiology and aetiology remain poorly understood. Available evidence suggests that circulating microRNAs (miRNAs) are associated with MDD, but it is still unknown whether miRNAs can predict subsequent incident MDD.In this nested case-control study, a total of 104 individuals, who were free of MDD at baseline, from the Women’s Health in Lund Area (WHILA) cohort were included. Among them, 52 individuals developed MDD (cases) during the 5 years follow-up and 52 individuals did not develop MDD (controls). Plasma expression levels of miR-17-5p, miR-134-5p, miR-144-5p, let-7b-5p and let-7c-5p at baseline were assessed using qRT-PCR. Logistic regression was used to estimate the odds of developing MDD among individuals with different levels of miRNA expression.p = 0.02) at baseline in cases compared to controls. After adjustment for age and BMI, let-7b-5p was negatively associated with odds for developing MDD . Moreover, let-7b-5p expression levels showed a trend over time with larger differences between cases and controls for the earlier cases (MDD diagnosis <2 years from baseline) than MDD cases developed later (MDD diagnosis 2–5 years from baseline).Plasma expression levels of let-7b-5p were significantly lower (These findings show that lower plasma levels of let-7b-5p are associated with a higher future risk of MDD. Results need to be validated in a large cohort to examine its potential as a peripheral biomarker for MDD.The online version contains supplementary material available at 10.1186/s12888-021-03621-4. Major depressive disorder (MDD) is a medical condition that includes abnormalities in mood, cognition, appetite, sleep, and psychomotor activity . MDD is Previous research concerning the pathophysiology of depression has mainly focused on genetic factors that affect the risk of development of depression. However, few genes were found to affect the risk of development of depression . More reThe most common ncRNAs are the microRNAs (miRNAs). MiRNAs are a class of endogenous non-coding single-stranded RNAs of approximately 21–23 nucleotides in length. MiRNAs are transcribed in the nucleus and transported to the cytoplasm where they can inhibit gene expression by either promoting messenger RNA (mRNA) degradation or by inhibiting translation through targeting of the 3′ untranslated region (UTR) of the target mRNA (Bartel ). MiRNAsOver the past decade, circulating miRNAs have already been identified as potential biomarkers in other diseases such as cancer and diabetes –12. In MThese results indeed indicate a difference in miRNA expression levels between patients and healthy controls, but they do not clarify whether they are the underlying cause or the consequence of MDD. Biomarkers, which are the consequence of MDD, can be a clear indicator of an MDD diagnosis. Instead, biomarkers that are the cause of MDD might be used as a predictive tool for the risk prediction of the development of MDD, as well as a therapeutic target for the treatment of MDD.The present study builds on the results of the aforementioned studies to determine whether a couple of miRNAs have the ability to predict the development of MDD. To our knowledge, this is the first study to investigate the prospective potential of miRNA expression in MDD. The study aimed to examine the association between miRNAs and the risk of MDD through the analysis of the expression levels (at baseline) of previously identified miRNAs  in a nested case-control study.n = 10,766) were invited to have their health status assessed. Participating women were included in the study from December 1, 1995, to February 3, 2000. A follow-up register study of the health care registers  at the Social Welfare Board on all individuals in the WHILA study (n = 6917) was performed until May 31, 2015. A total of 818 individuals were diagnosed with MDD. The diagnoses of MDD in WHILA were selected from the registers and based on the International Classification of Diseases with ICD-codes (ICD7-ICD10): F32–33, 296B, 298A, 311, 302, 296.00, 298.00.Study subjects were selected from the Women’s Health in Lund Area (WHILA) cohort Fig. ; a cohorWe excluded patients with prevalent cancer, prevalent CVD, prevalent diabetes (Type I and Type II), and prevalent mental disorder because these comorbidities were reported to be associated with the miRNAs selected in the present study –21.n = 188), and controls were randomly selected from subjects without MDD or other mental disorders during follow-up (n = 4058). It was reported previously that miRNAs seem to be more closely associated with early events (diseases occurring 0–5 years from blood sampling) compared to later events (diseases occurring 6–10 years from blood sampling) [Cases were randomly selected from subjects with the first diagnosis of MDD within 5 years after the baseline (ampling) . TherefoPrior to the study, we performed a power calculation using previous results based on the expression of miR-144-5p . This remiR-425-5p - Ct miR of interest. Normalisation stability of miR-425-5p was assessed in a previous study [Plasma samples were collected at baseline as described in a previous paper . Total Rus study .To ensure reproducibility of the samples, spike-in RNA was added during the RNA extraction and the cDNA synthesis. Both processes were assessed as reproducible as they had a coefficient of variation of 2.0 and 0.8%, respectively.The quality of plasma samples was assessed through the measurement of haemolysis. Samples were considered to have haemolysis when the difference in Ct-value between miR-23a-3p and miR-451a was bigger than 7.0 . Three sThis study was performed in-line with the principles of the Declaration of Helsinki. Sample storage and handling procedures were approved by the Lund/Malmö ethics committee . Written informed consent was obtained from all subjects.p < 0.05. All statistical analyses were performed in STATA version 16 (StataCorp LP).Characteristic variables for all individuals were presented. Data were presented as the mean and standard deviation (SD) for age and body mass index (BMI) whereas smoking and alcohol use were presented as a percentage. Both parametric and non-parametric tests were performed to test the difference in miRNA expression levels at baseline between case and control samples. Pearson’s correlation test was used to examine the correlation between miRNAs and other available peripheral blood biomarkers that we had measured. The results of the Student’s t-test were used for further analysis of normally distributed miRNAs - miR-134-5p, miR-144-5p, and let-7b-5p - whereas results of the Wilcoxon rank-sum test were used for miRNAs with a skewed distribution. Logistic regression was used to examine the association between cases and controls for miRNA expression levels at baseline. We adjusted for age and BMI. Trends in miRNA expression levels over time were assessed via trend analysis of groups dichotomised over time until MDD. Results were deemed significant when 2. Most women were non-smokers or low consumers of alcohol. There was no statistical difference between cases and controls in any of the confounders.Clinical characteristics of both cases and controls at baseline are shown in Table p = 0.02). For other miRNAs, significance was not reached but a trend of lower levels at baseline in cases compared to controls could be observed for miR-134-5p, miR-144-5p, and let-7c-5p. Levels at baseline in cases were higher for miR-17-5p than in controls, but not significant.During further analysis, a total of five samples were excluded due to poor quality of RNA, resulting in an analysis of 50 cases and 49 controls. MiRNA expression levels were measured for the five selected miRNAs. As shown in Table Correlations between let-7b-5p and other peripheral blood biomarkers such as triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and glucose were also analyzed. No correlations between let-7b-5p and the peripheral blood biomarkers were found Table .p = 0.03, 95% CI = 0.12–0.91). The results remained the same after adjustment for age and BMI.Logistic regression was used to estimate the odds ratio for having MDD among individuals with different levels of the selected miRNAs Table . The resp = 0.07).Finally, the expression levels of the five miRNAs were analysed with respect to the time interval between the date of blood sampling and the date of MDD diagnosis. It showed that let-7b-5p exhibited a trend over time Fig. , with laTo our knowledge, this is the first study investigating the association of circulating miRNA and the risk of MDD development. The main finding of this study was that let-7b-5p expression levels at baseline were significantly lower in plasma of individuals, who later developed MDD, compared to those who did not develop MDD. This suggests that let-7b-5p levels have potential as a circulating predictive biomarker in the risk assessment of the development of MDD. Other selected miRNAs had no significant difference in expression levels between cases (MDD) and controls (non-MDD).Many major cellular functions such as development, differentiation, growth, and metabolism are known to be regulated by miRNAs. As miRNAs play a major role in developmental processes, dysregulation of miRNA has been shown to be responsible for the development of numerous diseases such as cancer, cardiovascular disease and neurodevelopmental disease. Since the discovery of circulating miRNAs, most researchers have reported the use of miRNAs as circulating biomarkers for the diagnosis or prognosis of diseases . More reMiRNAs have dynamic roles in neurogenesis . TherefoMiRNA levels in peripheral blood reflect the miRNA levels in the central nervous system (CNS) as miRNA and other circulating nucleic acids can cross the blood-brain barrier (BBB) under physiological conditions . PatholoC. elegans by Reinhart et al. [In the present study, let-7b-5p was found to be negatively associated with the risk of MDD development. Let-7b-5p is part of the let-7 family. The let-7 family of miRNAs was first identified as a regulator of developmental timing in t et al. . Let-7 it et al. .Gururajan et al.  found a Let-7b-5p has been identified as a possible positive regulator of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway . ERK1/2 This study is the first nested case-control study to explore potential biomarkers for the risk prediction of MDD development. The results of this study were promising, but the limitations of the study must be kept in mind when evaluating the results. Firstly, the sample size was relatively low. The study design, though, required a smaller minimum sample size than what was used in the study. Secondly, cases and controls were unmatched in the study design. This, however, was corrected via adjustments in age and BMI in the statistical analysis. In addition, family history is a major factor concerning the risk assessment of MDD development , 35 The In summary, the findings of this study show that plasma levels of let-7b-5p have the potential as a biomarker of risk prediction for MDD development. Future studies are needed to validate and confirm the results in different cohorts with larger sample sizes.Additional file 1: Table S1. Correlation between peripheral blood biomarkers and let-7b-5p expression levels.